Assessing Liquid Inoculant Formulation of Biofertilizer (Sinorhizobium meliloti) on Growth, Yield, and Nitrogen Uptake of Lucerne (Medicago sativa)
Round 1
Reviewer 1 Report
1. Most of the abstract is the content of the background and materials and methods, please add the part of the results.
2. Citing references needs to be standardized in accordance with the requirements of the article.
3. Line 62, the scientific hypothesis of the article needs to be written clearly, which can be written in several points.
4. The meaning of bwwh in line 85 is not clear.
5. The literature standards in lines 94 and 96 are not accurate, and specific circumstances need to be written.
6. Nitrogenase activity is an important indicator for evaluating nitrogen-fixing bacteria. Have you measured it?
7. Please modify the format of the figure strictly according to the format of the published article.
8. Inoculation of rhizobia can increase the vigor of seeds, is there any physiological index for evaluation?
9. Why only one kind of rhizobia was used in the research, but not many kinds were selected for comparison?
10. In the field experiment, can the growth of lucerne be satisfied only by adding rhizobia without applying nitrogen fertilizer?
11. The discussion section needs to increase the number of references to improve the content of your research.
Author Response
Reviewer 1:
Comment 1: Most of the abstract is the content of the background and materials and methods, please add the part of the results.
-Response: The authors have addressed the above concerns in the manuscript.
Comment 2: Citing references needs to be standardized in accordance with the requirements of the article.
-Response: The authors have addressed the above concerns in the manuscript.
Comment 3: Line 62, the scientific hypothesis of the article needs to be written clearly, which can be written in several points.
-Response: The Hypothesis was added
Comment 4: The meaning of bwwh in line 85 is not clear.
-Response: According to bioclimatic conditions in South Africa (Climatic map for South Africa soil classification). NorthWest Province/Hartbeesfontein soil is classified as (Bwwh) according to Koppen classification index.
Comment 5: The literature standards in lines 94 and 96 are not accurate, and specific circumstances need to be written.
-Response: Refer to reference [16].
Comment 6: Nitrogenase activity is an important indicator for evaluating nitrogen-fixing bacteria. Have you measured it?
-Response: Indeed, nitrogenase activity is an important indicator for evaluating nitrogen fixation bacteria. We did not measure it. Our object was to measure the nodules as they fix atmospheric nitrogen and convert it into ammonia, the plant can utilize for growth and development. To meet the objective, the following parameters were measured: nodules numbers, nodules sizes and nitrogen content in plants.
Comment 7: Please modify the format of the figure strictly according to the format of the published article.
-Response: The reference to the tables were removed as per the editor recommendations, however we have deleted the tables and left the reference to the previous paper.
Comment 8: Inoculation of rhizobia can increase the vigour of seeds, is there any physiological index for evaluation?
Response: Yes, they are physiological index which explains the mechanism that foster seed vigour. Phytohormones such as IAA are well known to improve seed vigour. More explanation has been included in the discussion.
Comment 9: Why only one kind of rhizobia was used in the research, but not many kinds were selected for comparison?
Response: we used one strain (Sinorhizobium Meliloti) to determine it prevalence on South African soils. However, the study aimed at comparing two different formulations (T1 and T2) of same rhizobia strain to the T3 treatment which is a registered product in South Africa.
Comment 10: In the field experiment, can the growth of lucerne be satisfied only by adding rhizobia without applying nitrogen fertilizer?
Response: Indeed, growth of Lucerne can be satisfied by solely using biofertilizer (rhizobia). This was also evident in our research. However, factors such as soil classification do influence the success of rhizobia inoculation.
Comment 11: The discussion section needs to increase the number of references to improve the content of your research.
Response: The authors have addressed this concern by discussion the underlining mechanisms resulting in seed vigour, growth, and development improvement in inoculated plants.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The subject of the manuscript corresponds to the subject of the journal. At the same time, the manuscript should be significantly improved before publication, especially the Discussion section. I would like to recommend major revision.
Below I will provide some specific comments regarding the manuscript.
L10-28: The abstract should be reduced and generalized. Please briefly describe the main results and patterns found in the study.
L28: Could you suggest a more specific conclusion?
L29: Keywords should not repeat the title of the manuscript
L63: What hypothesis or hypotheses did you test in this study?
L89: Why was this soil layer chosen for analysis?
L90: Where tables 1 and 2 in the text?
L196: Were these differences statistically significant?
L208: What is the possible mechanism underlying the observed phenomenon?
LL 214, 217, 222, 224: What was the reason? What are the possible mechanisms underlying the observed phenomena?
LL 238-242: The conclusion needs to be improved. What is the fundamental significance of this study for science? What is its special interest for readers?
Author Response
Reviewer 2:
General comment: the authors have edited the language extensively.
Comment 1: L10-28: The abstract should be reduced and generalized. Please briefly describe the main results and patterns found in the study.
-Response: The abstract has been adjusted based on the three reviewers’ comments.
Comment 2: L28: Could you suggest a more specific conclusion?
-Response: The authors have suggested specific conclusions.
Comment 3: L29: Keywords should not repeat the title of the manuscript
-Response: The authors have changed the keywords accordingly.
Comment 4: L63: What hypothesis or hypotheses did you test in this study?
-Response: The authors have expounded on the hypothesis tested.
Comment 5: L89: Why was this soil layer chosen for analysis?
-Response: The land used were all flat land. The assumption made stated that there was no leaching of nutrients below 40 cm depth. In addition, the fact that lands for both climatic zones were fallow land for 5 years implies there was no nutrients content below 40cm depth.
Comment 6: L90: Where tables 1 and 2 in the text?
-Response: We were asked by editor to reference the first article where we used the same tables. So please see [16].
Comment 7: L196: Were these differences statistically significant?
-Response: We expressed the difference between the treatment in percentages.
Comment 8: L208: What is the possible mechanism underlying the observed phenomenon?
-Response: The authors have elaborated on the mechanisms involved in the processes.
Comment 9: LL 214, 217, 222, 224: What was the reason? What are the possible mechanisms underlying the observed phenomena?
-Response: The authors have elaborated on the mechanisms involved in the processes.
Comment 10: LL 238-242: The conclusion needs to be improved. What is the fundamental significance of this study for science? What is its special interest for readers?
-Response: The authors have addressed the comment
Author Response File: Author Response.docx
Reviewer 3 Report
The manuscript entitled "Assessing Liquid inoculant of Biofertilizer (Sinorhizobium Meliloti) on Growth, Yield and Nitrogen uptake of Lucerne (Medicago sativa)" is important for agricultural production and environmental issues. The experimental design and the results are good, but the following points should be revised for publication.
Title: Please revise the title as "Assessing the Effect of Liquid inoculant Dosage of Biofertilizer (Sinorhizobium Meliloti) on Growth, Yield and Nitrogen Accumulation of Lucerne (Medicago sativa)".
Lucerne is a nitrogen-fixing plants, so the N accumulated in plants derived from both N2 fixation in nodules and N absorption from roots. N uptake means only N absorbed by the roots and not include N derived from N fixation. In this research N from N2 fixation and N from N abosorption has been included.
Abstract: Line 20: Please change "6.5 x 109 (6.5 billion) viable cells g-1 " to "6.5 x 109 (6.5 billion) viable cells ml-1".
Line 27: Please change "uptake" to "accumulation".
Introduction: The references did not present in the Nitrogen format.
Line 33: [ 1]; [2]. is [1,2].
Line 50; Then the fixed N is further mineralised to supply organic N compounds. The term "mineralised" is misused. "assimilated" may be better.
Line 51-52. The sentence "The N fixation process is important for lucerne plant 51 due to its high N fertilizer requirement" is strange. Please change to "The N fixation process is important for lucerne plant 51 due to its high N fertilizer requirement".
Materials and Methods:
Line 76; "liquid inoculant contained Sinorhizobium meliloti 6.5 x 109 (6.5 billion) viable cells g-1 in the liquid inoculant" should be changed to "Liquid inoculant contained Sinorhizobium meliloti 6.5 x 109 (6.5 billion) viable cells ml-1 in the liquid inoculant.
Line 87: Please add the annual rain fall and the average temperature in both sites.
Line 88: "2.3. Analysis of Soil and fertilizer application" should be changed to "2.3. Analysis of Soil and biofertilizer application", because the authors did not apply chemical fertilizers as described in line 92.
Line 99-101 "The used treatments were Liquid inoculant dosages for Sinorhizobium meliloti (T0 = 0, T1 = 150, T2 = 300 and T3), 150 ml was also used for a registered standard. Inoculant was applied onto 101 50 kg of Lucerne seeds" lacks the dosage of treatment T3. Also please add ml for T0 = 0 ml, T1 = 150 ml, T2 = 300ml, and T3 ml for 50kg of seeds.
Line 114: plant length should be changed to plant hight.
Line 118: "Number and size of nodules were performed " should be changed to "Number and size of nodules were determined". Please add how to measure the size, such as the vertical length of the cylindrical nodule.
Line 124: "was used for nitrogen uptake" to "was used for determination of nitrogen concentration".
Line 194: This is not Nitrogen uptake. The authors measured the Nitrogen concentration (g N/kg DW).
Line 202-208: I cannot agree with this discussion, because inoculation of rhizobia may not influence the emergence rate, and the results of all treatments shown in Figure 1 were almost 100% and not so different.
Line216-217: I cannot understand this part.
Line 232: "Nitrogen uptake" to "Nitrogen concentration".
I recommend showing Nitrogen contents in plants per area (kg N/ha or gN/m2) instead of N concentration.
Author Response
Reviewer 3:
Comment 1: Abstract: Line 20: Please change "6.5 x 109 (6.5 billion) viable cells g-1 " to "6.5 x 109 (6.5 billion) viable cells ml-1".
-Response: The authors have amended,
Comment 2: Line 27: Please change "uptake" to "accumulation".
-Response: The authors have amended.
Comment 3: Introduction: The references did not present in the Nitrogen format.
-Response: The authors have addressed the comment.
Comment 4: Line 33: [ 1]; [2]. is [1,2].
-Response: The authors made the correction.
Comment 5: Line 50; Then the fixed N is further mineralised to supply organic N compounds. The term "mineralised" is misused. "assimilated" may be better.
-Response: The authors have modified to assimilated.
Comment 6: Line 51-52. The sentence "The N fixation process is important for lucerne plant 51 due to its high N fertilizer requirement" is strange. Please change to "The N fixation process is important for lucerne plant 51 due to its high N fertilizer requirement".
-Response: The authors made the correction.
Comment 7: Line 76; "liquid inoculant contained Sinorhizobium meliloti 6.5 x 109 (6.5 billion) viable cells g-1 in the liquid inoculant" should be changed to "Liquid inoculant contained Sinorhizobium meliloti 6.5 x 109 (6.5 billion) viable cells ml-1 in the liquid inoculant.
-Response: The authors made the correction.
Comment 8: Line 87: Please add the annual rain fall and the average temperature in both sites.
-Response: Refer to reference [16]
Comment 9: Line 88: "2.3. Analysis of Soil and fertilizer application" should be changed to "2.3. Analysis of Soil and biofertilizer application” because the authors did not apply chemical fertilizers as described in line 92.
-Response: The authors made the change.
Comment 10: Line 99-101 "The used treatments were Liquid inoculant dosages for Sinorhizobium meliloti (T0 = 0, T1 = 150, T2 = 300 and T3), 150 ml was also used for a registered standard. Inoculant was applied onto 101 50 kg of Lucerne seeds" lacks the dosage of treatment T3. Also please add ml for T0 = 0 ml, T1 = 150 ml, T2 = 300ml, and T3 ml for 50kg of seeds.
-Response: The comments were addressed in the article and the ml were added into dosages used.
Comment 11: Line 114: plant length should be changed to plant hight.
-Response: The authors made the change.
Comment 12: Line 118: "Number and size of nodules were performed " should be changed to "Number and size of nodules were determined". Please add how to measure the size, such as the vertical length of the cylindrical nodule.
-Response: The suggested phrase was added.
Comment 13: Line 124: "was used for nitrogen uptake" to "was used for determination of nitrogen concentration".
-Response: The authors took into consideration the comment and adjust to nitrogen content.
Comment 14: Line 194: This is not Nitrogen uptake. The authors measured the Nitrogen concentration (g N/kg DW).
-Response: The author adjusted to nitrogen content.
Comment 15: Line 202-208: I cannot agree with this discussion, because inoculation of rhizobia may not influence the emergence rate, and the results of all treatments shown in Figure 1 were almost 100% and not so different.
-Response: The emergence percentage in all treatments were higher due to the quality of the seed
Comment 16: Line216-217: I cannot understand this part.
-Response: The authors are not sure what is the issue.
Comment 17: Line 232: "Nitrogen uptake" to "Nitrogen concentration".
I recommend showing Nitrogen contents in plants per area (kg N/ha or gN/m2) instead of N concentration.
-Response: The authors changed to nitrogen content (g/kg )
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Accept in present form.
Author Response
Response: No comments
Reviewer 2 Report
The majority of previous comments was taken into account. I tend to think that the manuscript can be accepted in present form.
Author Response
Response 2: No comments received from reviewer 2 for the second report round 2. The reviewer accepted the manuscript in the present form. We thank the reviewer 2 for this work.
Reviewer 3 Report
The manuscript has been well revised, but still needs some corrections.
Please be careful to revise them.
Lines 118-119: Sinorhizobium meliloti (T0 = 0 ml, 118 T1 = 150 ml, T2 = 300 ml and T3 = ml), 150 ml was also used for a registered standard).
I think T3 should be 450 ml or 600 ml instead of 150 ml. Please be careful about T3.
Line 47: rasses [4-5] is [4,5].
Line 66: "Rhizobium S. meliloti" is "Sinorhizobium meliloti"
Line 75: Liquid inoculant of Biofertilizer (S.inorhizobium Meliloti) will have significant 75 impact on Growth, nodulation, Yield and Nitrogen contentuptake of Lucerne.
Liquid inoculant of biofertilizer (Sinorhizobium meliloti) will have significant impact on the growth, nodulation, yield, and nitrogen content of lucerne.
Line 81: Strain RF14 (Sinorhizobium meliloti)
Sinorhizobium meliloti, strain RF14
Author Response
Reviewer 3:
Comment 1: Lines 118-119: Sinorhizobium meliloti (T0 = 0 ml, 118 T1 = 150 ml, T2 = 300 ml and T3 = ml), 150 ml was also used for a registered standard).
-Response: The treatments used are (T0 = 0 ml, T1 = 150 ml, T2 = 300 ml and T3 = 150ml).
T3 is comparative registered product in South Africa.
Comment 2: Line 47: rasses [4-5] is [4,5].
-Response: Yes Line 47 is [4,5] and it was corrected.
Comment 3: Line 66: "Rhizobium S. meliloti" is "Sinorhizobium meliloti"
-Response: This is the common style for writing. Once the strain is written above in full name then we don’t need to repeat the whole name of the strain.
Comment 4: Liquid inoculant of Biofertilizer (S.inorhizobium Meliloti) will have significant 75 impact on Growth, nodulation, Yield and Nitrogen contentuptake of Lucerne.
-Response: The study aim was to assess the impact of Liquid inoculant formulation of Biofertiliser (S. Meliloti) on Growth, nodulation, Yield and Nitrogent content of Lucerne.
Comment 5: Liquid inoculant of biofertilizer (Sinorhizobium meliloti) will have significant impact on the growth, nodulation, yield, and nitrogen content of lucerne.
-Response: We added the suggested comments.
Comment 6: Line 81: Strain RF14 (Sinorhizobium meliloti) to change to Sinorhizobium meliloti, strain RF14
-Response: The change was addressed
Regards,
Auges Gatabazi
University of Pretoria
Department of Plant and Soil Sciences
Pretoria, 0002.
